Sub-project #1 Multiplex ELISA analysis of cell secretions Multiplex ELISA improves upon the traditional ELISA format. Rather than detecting a single antigen in a single sample, multiplex technology allows for the detection of up to 16 antigens from a single sample. This reduces the overall time and cost associated with ELISA and requires far less sample volume to produce more information. The multiplex format is customizable to allow researchers to investigate a unique array of proteins and peptides. Studies using multiplex ELISA include: -Analysis of mouse plasma samples from mice involved in social defeat models -Analysis of human plasma samples from subjects involved in sleep stimulation trials -Analysis of cell secretion products from Hela cells stimulated by UV light and cold. -Analysis of cell secretion products from Human Bronchial Epithelial cells irradiated X-rays -Analysis of primary microglial cells, treated with pro-inflammatory agent LPS. Sub-project #2 MALDI imaging of tissue samples to identify novel proteins MALDI-TOF Imaging is an emerging tool for the label free measurement of peptides, proteins, lipids, drugs and metabolites. The tissue can be imaged directly be the MALDI-TOF and combined with other modalities to study the molecular profiles as well as spatial placement of the tissue samples. Currently most tissue samples are imaged by MALDI and compared to histological stained tissues. In collaboration with NIAID, we are developing protocols to image tissues and then compare them to confocal microscopy/histo-cytometry imaged sections. The confocal microscopy imaging provides cellular level of resolution and spatial mapping whereas MALDI-IMS provides more high-throughput proteomics and molecular mapping that can identify biomolecules associated with tissue inflammation and other processes. The goal is to use these techniques to image lymph nodes infected with HIV in hopes of identifying new biomarkers and providing spatial resolution of them. In collaboration with LDR, we are developing protocols to image mice tissue that has been irradiated with ultrasound. We are imaging tissues pre and post treatment to determine if any proteins, peptides or lipids are unregulated by this procedure and identify them if they are. The goal of is to determine if the mice exhibit an inflammatory response to the procedure. Sub-project #3 Biacore Biacore 3000 detection system uses Surface Plasmon Resonance (SPR) to monitor the refractive index (RI) change as molecules interact at the sensor chip surface. SPR is the excitation of surface plasmons by light. One of the interactants (ligand) is immobilized on the sensor chip surface, while the other (analyte) is injected in continued flow over the surface. If the interaction takes place, the surface concentration of the analyte is increased that results in the RI change. SPR technology enables label-free real-time detection and monitoring of biomolecular events and provides qualitative and quantitative information on specificity of binding between two molecules, affinity, and kinetics of the interaction. Experimental design is highly flexible and the technology can be applied to protein interaction with other proteins, nucleic acids, lipids, small molecules. Studies using Biacore include: -Analysis of the binding kinetics of RNAse H2 toward a single ribonucleotide embedded in DNA duplex -Analysis of the membrane interactions of RasGRP1 and RasGRP3 with lipids to determine the role of different domains in binding interactions Sub-Project #4 Trace metal analysis by Inductively Coupled Plasma (ICP) ICP analysis with UV/Vis detection can be used to analyze samples for the presence of trace metals. The samples are introduced into a plasma flame and then detected by a continuous wavelength spectrophotometer to allow for quantitative measurements of the metal of interest. This technique allows researchers to determine yields for reactions, measure cellular uptake of metal compounds or determine metal levels in tissues in contrast agent experiments. Studies using ICP-OES include: - Analysis of magneto-plasmonic Janus vesicles integrated with Gold nanoparticles - Analysis of tissue samples for the presence of Boron nanoparticles - Analysis of multimodal contrast agents to quantify the amount of iron and gadolinium present - Analysis of novel drug eluting beads to quantify the amount of boron present - Analysis of poly(acrylic acid) gels to quantify the amount of sodium, calcium, potassium and chlorine present. Sub-project #5 High-throughput analysis of IgG glycosylation by matrix assisted laser desorption/ionization (MALDI) -time-of-flight (TOF)- mass spectrometry (MS). The Laboratory of Immunoregulation at NIAID focuses its research efforts on the elucidation of cellular and molecular mechanisms of the regulation of the human immune response in health and disease. A major component of these research efforts involves understanding the immunopathogenesis of B cells in HIV infection. Dr. Susan Moir of the B-Cell Immunology Unit has recently described a role for IgG3 in regulating B cells in HIV-infected individuals enrolled in clinical research protocol 02-I-0202. The findings indicated that glycosylation of IgG3 and possibly other serum proteins played a role in the regulatory function described in the study; the findings were obtained from the analysis of specimens isolated from over 100 participants. The evaluation of glycosylation patterns in serum proteins must be performed using an approach that is compatible with processing a relatively large number of samples from a relatively small quantity of material. Preliminary assays will be performed on a few positive and negative controls, followed by a few longitudinal samples from individuals who either begin with a positive IgG3 profile and then lose it over time or acquire the profile over time.